Total internal reflection ("TIR") is known in the art and is described with reference to FIG. 1. TIR operates upon the principle that light 10 traveling in a denser medium 12 (i.e. having the higher refractive index, N.sub.1) and striking the interface 14 between the denser medium and a rarer medium 16 (i.e. having the lower refractive index, N.sub.2) is totally reflected within the denser medium 12 if it strikes the interface at an angle, .theta..sub.R, greater than the critical angle, .theta..sub.C, where the critical angle is defined by the equation: EQU .theta..sub.0 C =arcsin (N.sub.2 /N.sub.1)
Under these conditions, an electromagnetic waveform known as an "evanescent wave" is generated. As shown in FIG. 1B, the electric field associated with the light in the denser medium forms a standing sinusoidal wave 18 normal to the interface. The evanescent wave penetrates into the rarer medium 16, but its energy E dissipates exponentially as a function of distance Z from the interface as shown at 20. A parameter known as "penetration depth" (d.sub.p - shown in FIG. 1A at 22) is defined as the distance from the interface at which the evanescent wave energy has fallen to 0.368 times the energy value at the interface. [See, Sutherland et al., J. Immunol. Meth, 74:253-265 (1984)] defining d.sub.p as the depth where E=(e.sup.-1).multidot.E.sub.0. Penetration depth is calculated as follows: ##EQU1##
Factors that tend to increase the penetration depth are: increasing angle of incidence, .theta..sub.R ; closely matching indices of refraction of the two media (i.e. N.sub.2 /N.sub.1 .fwdarw.1); and increasing wavelength, .lambda.. For example, if a quartz TIR element (N.sub.1 =1.46) is placed in an aqueous medium (N.sub.2 =1.34), the critical angle, .theta..sub.C, is 66.degree. (=arcsin 0.9178). If 500 nm light impacts the interface at .theta..sub.R =70.degree. (i.e. greater than the critical angle) the d.sub.p is approximately 270 nm.
Within the penetration depth, the evanescent wave in the rarer medium (typically a reaction solution) can excite fluorescence in the sample. This phenomenon has been used in the art with respect to immunoassays Harrick, et al., Anal. Chem., 45:687 (1973). Devices and methods that use TIR fluorescence for immunoassays have been described in the art by Hirschfield, U.S. Pat. Nos. 4,447,564, 4,577,109, and 4,654,532; Hirschfield and Block, U.S. Pat. Nos. 4,716,121 and 4,582,809, and U.S. Ser. No. 07/863,553 published as WO 93/20240 (Abbott Labs), which are all incorporated herein by reference. An immunospecific agent is adhered to the surface of the element and allowed to react with fluorescently labeled specific binding partners in the rarer medium. The specific binding results in the fluorescent labels being bound within the penetration depth. The emitted fluorescence (at the shifted wavelength) tunnels back into the TIR element, propagates within the TIR element along the same path as the standing sinusoidal wave (but at a different wavelength) and is detected at the output of the element.
TIR has also been used in conjunction with light scattering detection in a technique referred to as Scattered Total Internal Reflectance ("STIR"). See, e.g., U.S. Pat. Nos. 4,979,821 and 5,017,009 to Schutt, et al and WO 94/00763 (Akzo N. V.). According to this technique, a beam of light is scanned across the surface of a TIR element at a suitable angle and the light energy is totally reflected except for the evanescent wave. Particles such as red blood cells, colloidal gold or latex specifically bound within the penetration depth will scatter the light and the scattered light is detected by a photodetection means. WO 94/00763 also describes scanning the light beam across several loci of specific binding members which are either (1) the same binding member at varying concentration to achieve a wider dynamic range, or (2) different binding members to test for different analytes in a multiplex format. Scanning the light beam across multiple sites and gathering scattered light at each one is a very time-consuming process.
In U.S. Pat. No. 4,608,344 to Carter, et al., an optical waveguide is employed as the TIR element. In one variation, multiple binding sites are arranged on the waveguide in specific lines or grids to create a diffraction grating pattern of scattered light. By then looking at only specific orders of scattered light, this techniques minimizes the scattering caused by surface imperfections and/or impurities such as dust particles. (see FIG. 14 and columns 17-19).
Practical use of the Carter and STIR devices is severely limited by the serious background scattering from particles in solution. This background limits the sensitivity of detection of bound particles associated with analyte. The poor performance was compensated by sophisticated electronics and optics that could discriminate the small amount of signal over the high background levels. Electronic and optic complexity result in very expensive systems.
Finally, U.S. Pat. No. 5,192,502 to Attridge, et al., teaches a device comprising parallel plates defining a cavity for receiving a sample fluid. One plate serves as a waveguide and the other is coated with a layer of a light absorbing material.
Other background art of interest include the disclosure of Drmanac, et al. U.S. Pat. No. 5,202,231 which describes a new technique for the generation of nucleic acid sequence information known as sequencing by hybridization (SBH). According to this technique, a solid phase containing bound thereto an array of oligonucleotides of known sequence is allowed to hybridize with labeled DNA from a sample. Thus, a single hybridization experiment allows examination of a large number of different sites on a DNA molecule. Diagnosis of several human genetic conditions such as Duchenne muscular dystrophy or cystic fibrosis will likely require the resolving power of an SBH type system to determine the mutation associated with the disease state in an accurate and cost effective manner. One particular implementation of the SBH method uses a large number of oligonucleotides immobilized in a high density two dimensional array. Such a device has been called a "DNA chip" analogous to the high density circuits produced by the electronics industry. A sample of unknown DNA is applied to the chip and the pattern of hybridization determined and analyzed to obtain sequence information. WO 92/10588 and WO 92/10092 (Affymax Technologies N. V.) contain similar disclosures, as well as a photolithographic method for manufacturing such chips.
Since the stringency conditions affect hybridization, fine differentiation and specificity can be obtained if stringency can be accurately controlled. Thus, melting curves could provide an additional dimension to the DNA chip system and allow better differentiation of closely related sequences, a concern in implementation of SBH technology. The ability to change temperature and, in real time, monitor the chip hybridization patterns would be of great utility, particularly where there is a wide variation in GC content. Livshits, et al. J. Biomol. Struct. & Dynamics, 11:783-795 (1994) describe a DNA sequencing technique where discrimination of perfect and imperfect hybridizations was possible in a system of gel immobilized DNA using radioactive or fluorescent labels. The gel was subjected to one-minute washes every 5.degree. C. to remove label associated with imperfectly hybridized DNA. The authors claim the gel was advantageous due to a higher capacity for immobilization and higher discrimination power than other surfaces. However, the need to wash excess label from the surface, as well as the relatively long time for scanning the entire surface to obtain a measurement, impose significant limitations. For example, if one minute is required to read an entire DNA chip array and a one minute wash is needed at each incremental temperature, then a high resolution melting curve (e.g. every 1.degree. C.) front 30.degree. to 70.degree. C. would require an hour. The temperature would have to be held constant for one minute at each incremental temperature until all spots on the chip are measured.
Also of interest is the disclosure of co-owned, co-pending U.S. application Ser. No. 08/140,383, filed Oct. 21, 1993 and entitled APPARATUS AND METHOD FOR DETECTING A TARGET LIGAND, incorporated herein by reference. This application describes the use of a charge-coupled device "CCD" camera and image handling software to image and detect specific binding target ligands arranged in spatially separated, multiple loci on a single solid phase.